The role of Ca2+ in insulin-stimulated glucose transport in 3T3-L1 cells

We have examined the requirement for Ca2+ in the signaling and trafficking pathways involved in insulin-stimulated glucose uptake in 3T3-LI adipocytes. Chelation of intracellular Ca2+, using 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), resulted in >95% inhibition of insulin-stimulated glucose uptake. The calmodulin antagonist, W13, inhibited insulin-stimulated glucose uptake by 60%. Both BAPTA-AM and W13 inhibited Akt phosphorylation by 70-75%. However, analysis of insulin-dose response curves indicated that this inhibition was not sufficient to explain the effects of BAPTA-AM and W13 on glucose uptake. BAPTA-AM inhibited insulin-stimulated translocation of GLUT4 by 50%, as determined by plasma membrane lawn assay and subcellular fractionation. In contrast, the insulin-stimulated appearance of HA-tagged GLUT4 at the cell surface, as measured by surface binding, was blocked by BAPTA/AM.. While the ionophores A23187 or ionomycin prevented the inhibition of Akt phosphorylation and GLUT4 translocation by BAPTA-AM, they did not overcome the inhibition of glucose transport. Moreover, glucose uptake of cells pretreated with insulin followed by rapid cooling to 4 degreesC, to promote cell surface expression of GLUT4 and prevent subsequent endocytosis, was inhibited specifically by BAPTA-AM. This indicates that inhibition of glucose uptake by BAPTA-AM is independent of both trafficking and signal transduction. These data indicate that Ca2+ is involved in at least two different steps of the insulin-dependent recruitment of GLUT4 to the plasma membrane. One involves the translocation step. The second involves the fusion of GLUT4 vesicles with the plasma membrane. These data are consistent with the hypothesis that Ca2+/cahnodulin plays a fundamental role in eukaryotic vesicle docking and fusion. Finally, BAPTA-AM may inhibit the activity of the facilitative transporters by binding directly to the transporter itself.

Characterization of a chemsensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier

Chemosensory proteins (CSPs) are ubiquitous soluble small proteins isolated from sensory organs of a wide range of insect species, which are believed to be involved in chemical communication. We report the cloning of a honeybee CSP gene called ASP3c, as well as the structural and functional characterization of the encoded protein. The protein was heterologously secreted by the yeast Pichia pastoris using the native signal peptide. ASP3c disulfide bonds were assigned after trypsinolysis followed by chromatography and mass spectrometry combined with microsequencing. The pairing (Cys(I)-Cys(II), Cys(III)-Cys(IV)) was found to be identical to that of Schistocerca gregaria CSPs, suggesting that this pattern occurs commonly throughout the insect CSPs. CD measurements revealed that ASP3c mainly consists of alpha-helices, like other insect CSPs. Gel filtration analysis showed that ASP3c is monomeric at neutral pH. Using ASA, a fluorescent fatty acid anthroyloxy analogue as a probe, ASP3c was shown to bind specifically to large fatty acids and ester derivatives, which are brood pheromone components, in the micromolar range. It was unable to bind tested general odorants and other tested pheromones (sexual and nonsexual). This is the first report on a natural pheromonal ligand bound by a recombinant CSP with a measured affinity constant.

The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains similar to16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.

Binder treatment and lubricant system for aluminium P/M

Organic binders are used in premixes for powder metallurgy applications to prevent dusting and segregation. This is a particular problem for aluminium powder metallurgy because the dust is a potential safety hazard. The binder must also burn out completely at low temperatures in an inert environment and not react with the metal powders. It is demonstrated that cellulose acetate, polyvinyl acetate and polyvinyl alcohol are effective dedusting agents but they react with the metal powders during sintering and decrease the sintered density. Paraffin wan is an effect dedusting agent that provided die wall lubricity, does not interfere with sintering and increases tensile strength and ductility.

Facilitation of renal autoregulation by angiotensin II is mediated through modulation of nitric oxide

Aims: This study was designed to investigate the influence of angiotensin II (Ang II) and nitric oxide (NO) on autoregulation of renal perfusion. Methods: Autoregulation was investigated in isolated perfused kidneys (IPRK) from Sprague-Dawley rats during stepped increases in perfusion pressure. Results: Ang II (75-200 pM) produced dose-dependent enhancement of autoregulation whereas phenylephrine produced no enhancement and impaired autoregulation of GFR. Enhancement by Ang II was inhibited by the AT(1) antagonist, Losartan, and the superoxide scavenger, Tempol. Under control conditions nitric oxide synthase (NOS) inhibition by 10 muM N-omega-nitro-L-arginine methyl ester (L-NAME) facilitated autoregulation in the presence of non-specific cyclooxygenase (COX) inhibition by 10 muM indomethacin. Both COX and combined NOS/COX inhibition reduced the autoregulatory threshold concentration of Ang II. Facilitation by 100 pM Ang II was inhibited by 100 muM frusemide. Methacholine (50 nM) antagonised Ang II-facilitated autoregulation in the presence and absence of NOS/COX inhibition. Infusion of the NO donor, 1 muM sodium nitroprusside, inhibited L-NAME enhancement of autoregulation under control conditions and during Ang II infusion. Conclusions: The results suggest than an excess of NO impairs autoregulation under control conditions in the IPRK and that endogenous and exogenous NO, vasodilatory prostaglandins and endothelium-derived hyperpolarizing factor (EDHF) activity antagonise Ang II-facilitated autoregulation. Ang II also produced a counterregulatory vasodilatory response that included prostaglandin and NO release. We suggest that Ang II facilitates autoregulation by a tubuloglomerular feedback-dependent mechanism through AT(1) receptor-mediated depletion of nitric oxide, probably by stimulating generation of superoxide.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria

Recently, two fresh water species, 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis', and one marine species, 'Candidatus Scalindua sorokinii', of planctomycete anammox bacteria have been identified. 'Candidatus Scalindua sorokinii' was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle-the anammoxosome-in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography-mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18-23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential. (C) 2003 Elsevier B.V All rights reserved.

A role for pectin in the control of cell expansion

Uptake of nutrients and water depends on the growth of roots through elongation of individual cells near the. root tip. Many of the numerous components of Type I primary cell walls, those of dicotyledons and monocotyledons other than grasses (Poaceae), have been determined, and many hypotheses have been proposed for the control of cell expansion. This important aspect of plant growth still needs elucidation, however. A model is proposed in which pectin, which occurs as a calcium (Ca) pectate gel between the load-bearing cellulose microfibrils and xyloglucan (XG) chains, controls the rate at which cells expand. It is considered that the increasing tension generated by the expanding cell is transmitted to interlocked XG chains and cellulose microfibrils. The resulting deformation of the embedded Ca pectate gel elicits the excretion of protons from the cytoplasm, possibly via compounds such as cell wall-associated kinases, that weakens the Ca pectate gel, permitting slippage of XG molecules through the action of expansin. Further slippage is prevented by deformation of the pectic gel, proton diffusion, and the transfer of residual tension to adjacent XG chains. Evidence for this model is based on the effects of pH, Ca, and aluminum (Al) on root elongation and on the reactions of these cations with Ca pectate. This model allows for genetic selection of plants and adaptation of individual plants to root environmental conditions.

Neural reflex regulation of arterial pressure in pathophysiological conditions : interplay among the baroreflex, the cardiopulmonary reflexes and the chemoreflex

The maintenance of arterial pressure at levels adequate to perfuse the tissues is a basic requirement for the constancy of the internal environment and survival. The objective of the present review was to provide information about the basic reflex mechanisms that are responsible for the moment-to-moment regulation of the cardiovascular system. We demonstrate that this control is largely provided by the action of arterial and non-arterial reflexes that detect and correct changes in arterial pressure (baroreflex), blood volume or chemical composition (mechano- and chemosensitive cardiopulmonary reflexes), and changes in blood-gas composition (chemoreceptor reflex). The importance of the integration of these cardiovascular reflexes is well understood and it is clear that processing mainly occurs in the nucleus tractus solitarii, although the mechanism is poorly understood. There are several indications that the interactions of baroreflex, chemoreflex and Bezold-Jarisch reflex inputs, and the central nervous system control the activity of autonomic preganglionic neurons through parallel afferent and efferent pathways to achieve cardiovascular homeostasis. It is surprising that so little appears in the literature about the integration of these neural reflexes in cardiovascular function. Thus, our purpose was to review the interplay between peripheral neural reflex mechanisms of arterial blood pressure and blood volume regulation in physiological and pathophysiological states. Special emphasis is placed on the experimental model of arterial hypertension induced by N-nitro-L-arginine methyl ester (L-NAME) in which the interplay of these three reflexes is demonstrable.

Literatura oral e performance : a identidade e a ancestralidade no Ticumbi de Conceição da Barra, ES

O presente trabalho analisa a importância da literatura oral na construção da identidade de comunidades remanescentes de quilombos da região Norte do Estado do Espírito Santo, conhecidas como Sapê do Norte. São observados cantares, enredo, melodia, performance corporal e verbal gravada e transcrita dos Bailes de Congos de São Benedito. O corpus evidencia uma série de tensões sociais que cercam a região das comunidades de Sapê do Norte, sendo possível perceber forte relação entre o patrimônio cultural, a ancestralidade e a questão territorial, assim como constantes processos de desterritorialização e hibridismo nos campos religioso, social e linguístico. Na performance, o tempo passado se faz presente em memórias de tradições africanas que são revividas e atualizadas no momento enunciativo, possibilitando aos sujeitos históricos participantes do Ticumbi a escrita de uma narrativa híbrida, simbólica e tradutora de sua identidade plural. A literatura oral, efetivada por intermédio da performance cultural e pela constante troca realizada com a audiência, acessa a tradição e a sabedoria ancestral na prática enunciativa. A oralidade revive e atualiza a sabedoria dos antepassados, sendo, ao mesmo tempo, engendrada por esse saber e difusora da tradição de matrizes africanas. É nesse espaço de tensões que a tradição e a ancestralidade cantadas nos versos dos Bailes de Congos se constituem como o local da cultura, engendrado por lutas e tradições. Local em que a riqueza, a sabedoria e o universo simbólico das comunidades compõem um patrimônio cultural imaterial a ser preservado, difundido e legitimado socialmente.

A alteridade em Clarice Lispector

Percebendo a presença do tema da alteridade na obra de Clarice Lispector, este estudo o analisa no romance A paixão segundo G. H. e no conto “A mensagem” com respaldo na filosofia existencialista, amparado em Jean-Paul Sartre, Simone de Beauvoir, Frantz Fanon e Søren Kierkegaard e no que hoje seria o desdobramento desse olhar da filosofia existencialista dedicado ao estudo do outro, desdobramento no qual privilegiaremos as reflexões de Pierre Bourdieu, Kwame Anthony Appiah e Jacques Derrida.

Caracterização da ontogenética do pau-brasil

As ações de reflorestamento com o pau-brasil (Caesalpinia echinata Lam.) depende de informações de suas características ecofisiológicas sujeitas às variações ontogenéticas e ambientais. O objetivo desse trabalho foi caracterizar alguns aspectos morfológicos, anatômicos, fisiológicos e estruturais de parede celular de C. echinata nas fases juvenil, jovem e adulto em condições naturais em um fragmento da Floresta Atlântica. Foi analisada a biometria, concentração de nutrientes e dos pigmentos cloroplastídicos dos foliólulos, anatomia foliar e do xilema secundário do caule e a constituição dos polímeros estruturais de parede celular. Os indivíduos juvenis localizados no estrato inferior da floresta se destacaram pela maior área foliar específica e maiores teores de pigmentos cloroplastídicos bem como pelas maiores dimensões de suas células guardas associado às maiores concentrações de K e Ca foliar. Estruturalmente, os indivíduos juvenis apresentaram menores elementos de vasos e teores de lignina. Os indivíduos jovens apresentaram valores intermediários das variáveis analisadas. Já os indivíduos adultos, cujas copas alcançavam o dossel, se destacaram pelo maior espessamento do limbo, da cutícula e do parênquima lacunoso, teor de água foliar, densidade estomática e teor de lignina foliar e caulinar cuja capacidade de síntese foi associada ao maior teor de P foliar. O conteúdo de celulose foliar e caulinar não variou entre as diferentes fases ontogenéticas. As hemiceluloses são do tipo xilanos com possibilidade de presença de xiloglucano dada a maior fração de xilose (±12% MS) e galactose (±1% MS). A glucose foi o monossacarídeo mais representativo (±40% MS) sem diferenças ontogenéticas. As diferenças morfológicas, anatômicas, fisiológicas e estruturais parecem, também, estarem sob controle da irradiância mais intensa na copa dos indivíduos adultos. Os resultados denotam que o plantio consorciado com espécies de crescimento rápido seja a melhor ação para o reflorestamento de C. echinata.

Sorriso de persona : estudos sobre teatro e recepção

Danilo Barcelos, Ester Abreu Vieira de Oliveira, Maria Mirtis Caser (orgs.).

Halitose: proposta de um protocolo de avaliação

A Halitose é um sintoma constrangedor com significativo impacto social. Ela afeta milhões de pessoas ao redor do mundo e muitos recursos são investidos em produtos para a melhora do hálito, sem sucesso. O estudo da halitose com uma abordagem científica se justifica, uma vez que a halitose é causa de restrição social, diminui a qualidade de vida e pode ser indicativo da presença de doenças mais graves. OBJETIVO: Elaborar um protocolo de avaliação da halitose com o objetivo de minimizar custos, evitar exames desnecessários e orientar o diagnóstico causal. MÉTODO: O protocolo foi elaborado baseado na literatura e na experiência pessoal dos autores, adotando uma anamnese baseada em evidências. RESULTADO: Existem muitas causas da halitose e a maioria delas está relacionada com a cavidade oral, outras estão relacionadas com doenças otorrinolaringológicas e respiratórias. Doenças gastrointestinais, alterações das funções renais e hepáticas e outras síndromes metabólicas são causas menos freqüentes, porém importantes de halitose. CONCLUSÃO: Existem custos importantes envolvidos no diagnóstico e tratamento da halitose, incluindo consultas ambulatoriais, avaliação de especialistas e exames complementares. Estes custos poderiam ser minimizados adotando uma anamnese baseada em evidências e um organograma para uma investigação clínica racional.